PKR activation and eIF2α phosphorylation mediate human globin mRNA splicing at spliceosome assembly

Abstract

Short elements in mammalian mRNA can control gene expression by activating the RNA-dependent protein kinase PKR that attenuates translation by phosphorylating cytoplasmic eukaryotic initiation factor 2α (eIF2α). We demonstrate a novel, positive role for PKR activation and eIF2α phosphorylation in human globin mRNA splicing. PKR localizes in splicing complexes and associates with splicing factor SC35. Splicing and early-stage spliceosome assembly on β-globin pre-mRNA depend strictly on activation of PKR by a codon-containing RNA fragment within exon 1 and on phosphorylation of nuclear eIF2α on Serine 51. Nonphosphorylatable mutant eIF2αS51A blocked β-globin mRNA splicing in cells and nuclear extract. Mutations of the β-globin RNA activator abrogated PKR activation and profoundly affected mRNA splicing efficiency. PKR depletion abrogated splicing and spliceosome assembly; recombinant PKR effectively restored splicing. Excision of the first intron of β-globin induces strand displacement within the RNA activator of PKR by a sequence from exon 2, a structural rearrangement that silences the ability of spliced β-globin mRNA to activate PKR. Thus, the ability to activate PKR is transient, serving solely to enable splicing. α-Globin pre-mRNA splicing is controlled likewise but positions of PKR activator and silencer are reversed, demonstrating evolutionary flexibility in how PKR activation regulates globin mRNA splicing through eIF2α phosphorylation.

Protein kinase R and its cellular regulators in cancer: An active player or a surveillant?

“…Early in vitro studies identified cellular RNAs with secondary structure as PKR regulators. These include 3 0 -untranslated region (UTR) of α-tropomyosin (Davis & Watson, 1996), stem-loop structure in the 3 0 -UTR of TNF-α mRNA (Osman, Jarrous, Ben-Asouli, & Kaempfer, 1999), 5 0 -pseudoknot structure of IFN-γ mRNA (Ben-Asouli, Banai, Pel-Or, Shir, & Kaempfer, 2002), and RNA activator elements of β-globin pre-mRNAs (Ilan et al, 2017) (Table 2). Although interesting, it should be recalled that their action on PKR is mostly based on in vitro data and their genuine cellular roles need to be validated.…”

“…Ben-Asouli et al, 2002;Cohen-Chalamish et al, 2009) β-Globin pre-mRNAs Activation Unknown(Ilan et al, 2017) …”

Induction of an alternative 5’ leader enhances translation of Inpp5e and resistance to oncolytic virus infection

“…The question remains how infection signals the switch to an alternative "ribosome-engaged" transcriptome? PKR-mediated splicing has been previously reported 74 ; however, we were unable to link PKR activation to our change in variant expression. Further studies will be required to home-in on the signalling axis that mediates increased expression of the short Inpp5e transcript.…”

“…4F). PKR activation has been previously shown to modulate mRNA splicing 47 ; therefore we asked if induction of the short Inpp5e variant was dependent on this kinase. Although we could re-capitulate the poly(I:C)-mediated induction of the short variant in wildtype mouse embryonic fibroblasts (MEFs), there was a similar induction in their PKR-null counterparts 48,49 , suggesting that the short variant induction is PKRindependent ( Fig.…”

Daphnia magna modifies its gene expression extensively in response to caloric restriction revealing a novel effect on haemoglobin isoform preference

“…RNA-dependent protein kinase R (PKR) is an intracellular sensor of stress that has recently been shown to also be essential for globin gene expression generally (Ilan et al, 2017). It could potentially also act in conditions of cellular stress to modulate haemoglobin expression, while in Drosophila knockdown of a globin gene expressed in the trachea reduces survival under hypoxia (Gleixner et al, 2016;Harrison et al, 2018), presumably due to a role in providing adequate oxygen levels.…”